Polysaccharide of Trametes robiniophila Murr inhibits the recruitment of myeloid-derived suppressor cells by modulating the JAK1/STAT3/CCL2 axis to suppress triple-negative breast cancer progression

Myeloid-derived suppressor cells (MDSCs) are frequently over-recruited in the immune microenvironment of triple-negative breast cancer (TNBC), forming an immunosuppressive barrier that contributes to the poor response rates to existing immunotherapies. This study aims to investigate the effects of polysaccharide of Trametes robiniophila Murr (PS-T) on the immune microenvironment in TNBC and clarifying the underlying molecular mechanism by which PS-T inhibits MDSC recruitment through suppression of the JAK1/STAT3/CCL2 axis.

TNBC xenograft model was established in 36 female BALB/c mice (6 to 8 weeks, weighing 18 to 20 g). Mice were randomly divided into 2 groups (n=6) for intragastric administration: PS-T group (100 µL solution containing 3 mg of PS-T) and control group (100 µL isotonic sodium chloride solution). Tumor volume was measured on days 5, 7, 9, 11, 13, 15, 17, 19, and 21 post-inoculation. On day 21, tumor tissues were harvested and photographed, and then processed into single-cell suspensions and tissue sections for flow cytometry and immunohistochemical staining to evaluate the infiltration of MDSCs and T cells. RNA sequencing data were analyzed using GO and KEGG enrichment analyses to identify signaling pathways involved in the PS-T-mediated MDSC regulation in TNBC. RT-qPCR, Western Blot, and immunohistochemical staining were used to detect JAK1, STAT3, and CCL2 expression. Transwell co-culture assay was performed to verify that PS-T inhibits MDSCs recruitment by suppressing CCL2 secretion. JAK1-knockdown 4T-1 cell line was constructed, and Western blot and ELISA were used to assess the effects of JAK1 knockdown on PS-T-mediated inhibition of phosphorylated STAT3 and CCL2.

① Compared with the control group, PS-T significantly inhibited the growth of TNBC (P=0.003) and remodeled the immune microenvironment. Flow cytometry showed that the proportion of intratumoral CD8⁺ T cells was increased in the PS-T group (P=0.002), which was confirmed by immunohistochemistry demonstrating elevated numbers of intratumoral CD8⁺ T cells (P=0.003). ② Single-cell RNA sequencing of the immune cell composition in tumor tissues from TNBC-bearing mice revealed that PS-T treatment increased CD8⁺ T cell infiltration from 3.6% to 9.33%, while markedly reducing the proportion of MDSCs from 15.64% to 0.17%. Immunohistochemical staining further showed a significant decrease in the number of CD11b⁺ cells in the PS-T group (P=0.008). Flow cytometric analysis of MDSC subsets indicated that PS-T significantly reduced the proportion of PMN-MDSCs (gated as CD45⁺CD11b⁺Ly6G⁺Ly6C^low cells) (P=0.012). ③ GO and KEGG enrichment analyses of differentially expressed genes between the control and PS-T groups revealed significant enrichment in cytokine-related pathways and the JAK/STAT signaling pathway. RT-qPCR and Western blot assays demonstrated that PS-T significantly downregulated the mRNA and protein levels of CCL2 in 4T-1 cells (P<0.001), which was further confirmed by immunohistochemistry showing reduced CCL2 expression in vivo (P<0.001). Co-culture of flow cytometry-sorted MDSCs with 4T-1 cells in Transwell chambers in the presence of MK-0821, an antagonist of the CCR2 receptor, attenuated the PS-T-mediated inhibition of MDSC recruitment to the lower chamber (P<0.01). ④ Both immunohistochemistry and Western blot analyses demonstrated that PS-T inhibited the phosphorylation of STAT3 (p-STAT3) without altering the total STAT3 protein level. ⑤ Western blot analysis showed that PS-T reduced the protein level of JAK1, the kinase responsible for STAT3 phosphorylation (P=0.003). Knockdown of JAK1 in 4T-1 cells abrogated the inhibitory effects of PS-T on p-STAT3 and CCL2 expression, as verified by both Western blot and ELISA assays.

PS-T suppresses intratumoral MDSC recruitment by inhibiting the JAK1/STAT3/CCL2 signaling pathway in TNBC tumor cells, thereby remodeling the immunosuppressive microenvironment and exerting anti-tumor effects. These findings provide novel theoretical support for the application of PS-T in combination immunotherapy for TNBC.